Effects of Loop Nucleobase Substitution on G-Quadruplex Thermal Stability in Aqueous Glycine Betaine, Proline, TMAO, and Urea Solutions

G-quadruplexes are guanine-rich DNA or RNA structures comprising two or more stacked guanine tetrads (G-quartet) with nucleobases in the loops linking the G-quartets. The thermal stability of the thrombin-binding aptamer G2 d(G2T2G2TGTG2T2G2) G-quadruplex was quantified using m-values in aqueous glycine betaine (GB), proline, TMAO, and urea solutions using UV-absorbance spectroscopy. The thermal stability of the G2 variants was also explored with nucleobase substitutions in the TGT loop (TAT, TCT, TTT, UGU, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). GB and TMAO strongly stabilized G2 and its variants. Urea weakly destabilized G2 and its variants, while proline acted as a weak stabilizer or destabilizer depending on G2 variant nucleobase sequence. Both the unfolding enthalpy and entropy were positively correlated with cosolute molality for all cosolute and G-quadruplex combinations. Analysis of the change in solvent-accessible surface area (ΔASA) after unfolding G2 suggested the stability of G2 and its variants in aqueous cosolute solutions was driven by favorable interaction of cosolutes with G-quartet ΔASA and net-favorable or unfavorable interactions with the loop ΔASA. Our results reinforce a general mechanism of folded DNA and RNA stability modulation in cosolute solutions.